Phenetic Analysis of Morphological and Molecular Traits in Acanthaceae Juss

http://dx.doi.org/10.4236/jbm.2015.33004

How to cite this paper:

Abdel-Hameed, U.K., Tantawy, M.E., Salim, M.A., Mourad, M.M. and Ishak, I.F. (2015) Phenetic

Analysis of Morphological and Molecular Traits in Acanthaceae Juss. Journal of Biosciences and Medicines, 3, 18-34.

http://dx.doi.org/10.4236/jbm.2015.33004

Phenetic Analysis of Morphological and

Molecular Traits in Acanthaceae Juss

Usama K. Abdel-Hameed

_{, Mohamed E. Tantawy, Mohamed A. Salim, Magdy M. Mourad,}

Ishak F. Ishak

Botany Department, Faculty of Science, Ain Shams University, Cairo, Egypt

Email:

_{[email protected]}

Received November 2014

Abstract

Acanthaceae has received considerable taxonomic attention at the familial, subfamilial, tribal and

subtribal levels. Several different infra-familial classifications have been proposed for the

Acan-thaceae, but no taxonomic consensus has yet been reached. The main objective of the present

study is to throw light on the phenetic relationships and to explore the contribution of

morpho-logical and molecular characters in systematics of Acanthaceae. The morphomorpho-logical data viz.

ma-cromorphology, stomatography, lamina architecture and ISSR profiles of 30 Egyptian

acantha-ceous taxa were investigated. The phenetic analysis using NTSYS-PC version 2.02 software based

on 55 potentially informative morphological and molecular characters indicated that the used

morphological and ISSR criteria is likely to be useful and valuable taxonomic traits. The

morpho-logical characters and ISSR aspects of all the studied species produced a phenogram that showed

two series; one of them had two subseries, the first one comprised only three taxa while the

second divided into two clusters, each contained two groups. The delimitation and the

member-ship of the studied taxa clearly merit additional study using more criteria. The phenetic analysis of

both morphological and molecular attributes clarified the segregation of genus Avicennia as a

dis-tinct identity away from Acanthaceae. Acanthus mollis & A. montanus are isolated in its own series

that comparable to tribe Acantheae of the current taxonomic systems. The studied species of

Thunbergia are gathered its own subseries that comparable to tribe Thunbergiae and Ruellia in its

own group that comparable to tribe Ruellieae.

Keywords

Phenetic Analysis, Numerical Taxonomy, Morphology, ISSR, Acanthaceae

1. Introduction

Acanthaceae has received considerable taxonomic attention at the familial, subfamilial, tribal and subtribal

le-vels by various workers, the earlier system of infra-familial classifications of Acanthaceae is that of [1], he

sub-divided the family on the basis of two to four stamens and recognized either genera viz.

Acanthus

,

Barleria

,

Blepharis

,

Dianthera

,

Dilvaria

,

Justicia

,

Ruellia

and

Thunbergia

. Since then, the family has been subjected to

various taxonomic treatments and has received attention from many authors; [2] recognized four subfamilies viz.

Nelsonioideae, Mendoncioideae, Thunbergioideae and Acanthoideae on the basis of types of fruits, number of

ovules and presence or absence of retinacula and their shape. He further subdivides Acanthoideae into two

sec-tions (Contortae and Imbricatae).

Several different infra-familial classifications have been proposed for the Acanthaceae, but no taxonomic

consensus has yet been reached [3]. On the basis of morphology it has been suggested that the family is not

“natural” [4] [5]. Molecular data has helped botanists move towards a more clearly circumscribed family, by

supporting the inclusion of

Avicennia

[6],

Thunbergia

and others that often receiving their own family status [7].

This has, however, led to a situation where the family cannot be definitively and distinctively constrained by

morphological characters [8]. Recent work on the evolution and the diversification of the Acanthaceae provides

a phylogenetic context for assessing the taxonomic significance of possible characters (including micro-mor-

phological structures) within the family [9].

Two subfamilies were recognized by [10]; Anechmatacantheae (without retincula) and Echmatacantheae

(with retinacula). The former comprises two tribes

viz

. Thunbergieae and Nelsonieae, which together include

[2]’s Thunbergioideae, Nelsonioideae and Mendoncioideae. The latter has nine tribes (Hygrophileae, Ruellieae,

Barlerieae, Acantheae, Aphelandreae, Gendarusseae, Eranthemeae, Dicliptereae and Andrographideae)

com-prising [2]’s Acanthoideae.

The family was split by [11] and [12] into three suborders viz.; Thunbergideae (with

Thunbergia

), Ruellideae

(with all the genera having contorted corolla lobes in bud) and Acanthideae (genera with imbricate corolla

lobes). Tribes and sub-tribes were recognized within last two suborders in this system; Nelsonieae is treated as a

tribe of Ruellideae, tribes Gendarussae and Dicliptcreae are amalgamated to form the tribe Justicieae with a

number of sub-tribes. The present family was classified by [13] into five tribes; Thunbergieae, Nelsonieae,

Ru-ellieae, Acantheae and Justicieae. The tribe Thunbergieae and Nesonieae correspond to [2]’s Thunbergioideae

and Nelsonioideae respectively while Acantheae, Ruellieae and Justicieae together constitute his Acanthoideae.

The number of tribes was reduced by [14] to a total of five with Thunbergieae being reduced to tribal level; the

Ruellieae, Nelsonieae and Acantheae remaining mostly unchanged. However in this system the tribe Justicieae

was expanded, comprising the previously recognized tribes of Barlerieae, Andrographideae and Asystaceae at

the sub-tribal level. Apart from the reduction in the rank of some tribes to sub-tribes, this classification is similar

to [11] [12]’s treatment.

For the first time Thunbergiaceae was raised as a separate family by [15] accommodating the first three

sub-families of Acanthaceae sensu [2], [13]’s Acanthaceae comprising [2]’s subfamily Acanthoideae only. Two

subfamilies were formulated by [16]; Thunbergioideae and Acanthoideae that correspond to [13]’s

Thunbergia-ceae and AcanthaThunbergia-ceae and [10]’s Anechmatacantheae and Echmatacantheae. [14]’s Thunbergioideae also

in-cludes the first three sub-tribes of [2]. [17] comprised five tribes and sixteen sub-tribes, this classification has

been accepted by many taxonomists and is in use even today [18]-[24]. In other works [25]-[28], it is well

ap-preciated, though with some modifications.

The extensive work of [5] was thrown much light on the classification of the family. According to him [2]’s

Thunbergioideae and Mendoncioideae show greater affinity to Bignoniaceae and Pedaliaceae than to latter’s

Acanthoideae; and his Nelsonioideae is very much related to the tribe Rhinantheae of Scrophulariaceae. In this

consideration Bremecamp suggested to place Nelsonioideae near Rhinantheae as long as the latter is placed in

Scrophulariaceae. But this reshuffling has received varying response. He was also recognized two subfamilies

viz

. Acanthoideae and Ruellioideae, the former with five tribes and the latter with seven tribes. Along with

pol-len characters [5] did use other supporting characters such as nature of corolla, fruits and seeds. But within

Jus-ticeae there are a number of genera with uncertain positions [5]. He proposed many changes in the generic

lim-its.

There have been some molecular systematic studies specifically addressing the higher level systematics of

Acanthaceae [3] [32]-[34]. The genes used for these studies were rbcL, ndhF, trnL-trnF and trnL-trnF combined

with ITS, respectively. The rbcL data of [32] was re-analyzed by [3] but provided limited resolution within

Acanthaceae. The strict consensus tree of [3] and [34] resolved Acanthaceae sensu [2] as a group. These

analys-es demonstrated that there is no support for separating

Thunbergia

from Acanthaceae as proposed by [5]. The

strict consensus tree of [33] resolved Acanthaceae sensu [2] with the exception of

Elytraria

(Nelsonioideae)

which remained unresolved. [35] showed that

Thunbergia

, are a natural group based upon the shared possession

of woody bristles on the anthers, lack of an endothecial cell layer, elongated connective tips and poricidal

open-ing of the thecae.

The main objective of the present study is to throw light on the phenetic relationships and to explore the

con-tribution of morphological and molecular characters in systematics of Acanthaceae.

2. Materials and Methods

2.1. Sampling

The present study was conducted on 30 species of Acanthaceae in Egypt belonging to 17 genera that were

col-lected from some Egyptian botanical gardens.

Blepharis edulis

and

Avicennia marina

were collected from

natu-ral habitats in Sinai Peninsula (

Table 1

). The identification of wild taxa was taken by the aid of [36] and [37],

while the horticultural taxa with the help of [38] and [39]. The taxa were further matched against the dried

spe-cimens in the Herbaria at Ain Shams University, Faculty of Science (CAIA), Cairo University, Faculty of

Science (CAI), Flora and Phytotaxonomy Research Department (CAIM) and Orman Botanical Garden (Geiza).

Voucher specimens of the studied species were kept in CAIA.

2.2. Morpholoical Investigation

Macromorphological characters were examined directly from the investigated specimens. Stomatography (LM

and SEM) was carried on the bases of traditional method of [40]. A Reichert Microstar IV microscope was used

for microphotographs documentation at the Plant Taxonomy Research Laboratory, Botany Department, Faculty

of Science, Ain Shams University, Cairo, Egypt. Lamina vein architecture was done according to [41].

Descrip-tive terminology of epidermal characteristics based on [41]-[44] while leaf architectural terminology follows

[41].

2.3. Molecular Investigation

Genomic DNA extraction was performed as suggested by Qigene multisource Genomic DNA Mini-Prep Kit

(USA, cat. No. Ap-MN-MS-GDNA-50). DNA samples of each plant were analyzed individually to detect intra-

specific variations and bulked to detect inter-specific variations. An initial screening of 20 ISSR primers

(suc-cessfully utilized in other plant species, was performed in order to test their readability and amplification

pro-files for polymorphism). After this screening procedure, five ISSR primers were selected (

Table 2

). Polymerase

chain reactions (PCR) were carried out according to [45]. The products of ISSR based PCR were detected by

electrophoresis on agarose gel (1.2% in 1× TBE buffer), then stained with ethidium bromide (0.3 ug/ml).

Am-plicon sizes were estimated using 1 Kb DNA standard (Bioron, Germany). Reproducible bands visualized on the

gels were scored using a binary code (1/0) for their presence or absence based on the UVP gel documentation

system (Gel Works ID advanced software, UVP).

2.4. Phenetic Analysis

Unweighted Pair-Group Method using Arithmetic Averages with SAHN function [46] was used to estimate

states of characters variation among the species, each taxa was considered as operational taxonomic unit (OTU)

and states of characters analysed as binary characteristics. The formation of groups was depending on the values

of similarity. All computations were carried out by the aid of the NTSYS-PC version 2.02 [47].

3. Results

Table 1.

The studied taxa of acanthaceae and avicennia marina.

1 Acanthus mollis L.-Sp. Pl. 2: 639. 1753 [1 May 1753] (IK)

2 A. montanus T. Anderson-J. Proc. Linn. Soc., Bot. 7: 37. 1863 [1864 publ. 1863] (IK)

3 Anisacanthus virgularis Nees-Prodr. [A. P. de Candolle] 11: 445. 1847 [25 Nov 1847] (IK)

4 Aphelandra squarrosa Nees-Fl. Bras. (Martius) 9: 89. (IK)

5 Barleria cristata L.-Species Plantarum 2 1753 (APNI)

6 B. prionitis L.-Sp. Pl. 2: 636. 1753 [1 May 1753] (IK)

7 Blepharis edulis (Forssk.) Pers.

8 Eranthemum nervosum R. Br. ex Roem. & Schult.-Syst. Veg., ed. 15 bis [Roemer & Schultes] 1: 174. 1817 [Jan-Jun 1817] (IK)

9 Fittonia argyroneura E. Coem.-Fl. des Serres xvi. (1865-67) 103. (IK)

10 F. verschaffeltii (Lem.) Van Houtte-Fl. des Serres 15: 186. 1865; cf. R.K.Brummitt in Curtis’s Bot. Mag., 182(4): 167. 1979 (IK)

11 Hypoestes sanguinolenta Hook-Bot. Mag. 91: t. 5511. 1865 (IK)

12 Jacobinia carnea (Lindl.) G. Nicholson-Ill. Dict. Gard. ii. 206 (1885) (IK)

13 Justicia adhatoda L.-Sp. Pl. 1: 15. 1753 [1 May 1753] (IK)

14 J. betonica L.-Sp. Pl. 1: 15. 1753 [1 May 1753] (IK)

15 J. brandegeeana Wassh. & L. B. Sm.-Fl. Ilustr. Catarin. Pt. 1, Acantac., 102 (1969) (IK)

16 J. gendarussa Burm.f.-Fl. Ind. (N. L. Burman) 10. 1768 [1 Mar-6 Apr 1768] (IK)

17 J. spicigera Schltdl.-Linnaea 7: 395. 1832 (IK)

18 Odontonema cuspidatum (Nees) Kuntze-Revis. Gen. Pl. 2: 494. 1891 [5 Nov 1891] (GCI)

19 Pachystachys lutea Nees-Prodr. [A. P. de Candolle] 11: 320. 1847 [25 Nov 1847] (IK)

20 Pseuderanthemum atropurpureum Radlk.-Sitzungsber. Math.-Phys. Cl. Königl. Bayer. Akad. Wiss. München 13: 286. 1884 ; nom. inval. (IK)

21 P. bicolor Radlk.-Sitzungsber. Math.-Phys. Cl. Königl. Bayer. Akad. Wiss. München 13: 286. 1884; nom. inval. (IK)

22 Ruellia alba Nees-Fl. Bras. (Martius) 9: 55 (IK)

23 R. brittoniana Leonard-J. Wash. Acad. Sci. xxxi. 96 (1941); Fernald in Rhodora, xlvii. 7 (1945),cum descr. ampl. et emend (IK)

24 R. glabra B.Heyne ex Roth-Nov. Pl. Sp. 312. 1821 [Apr 1821] (IK)

25 R. humilis Nutt.-Trans. Amer. Philos. Soc. ser. 2, 5: 182. 1835 [late 1835] (IK)

26 Sanchezia nobilis Hook. f.-Bot. Mag. 92: t. 5594. 1866 (IK)

27 Thunbergia alata Bojerex Sims-Bot. Mag. 52: t. 2591. 1825 (IK)

28 T. erecta T. Anderson-J. Proc. Linn. Soc., Bot. 7: 18. 1863 [1864 publ. 1863] (IK)

29 T. grandiflora Roxb.-Edwards’ Botanical Register 6 1820 (APNI)

30 Avicennia marina (Forssk.) Vierh.-Denkschr. Kaiserl. Akad. Wiss., Wien. Math.-Naturwiss. Kl. lxxi. 435 (1907) (IK)

Table 2.

ISSR primer names, sequence, and annealing temperature (Ta).

No. Primer Name Sequence Ta (˚C)

1 890 ACG (GT)7 50˚C

2 17898b (CA)6 GT 40˚C

3 835 (AG)8 CC 55˚C

4 851 (GT)8 CG 55˚C

all the studied species were summarized in

Tables A1-A4

. The profiles of ISSR that illustrated in

Figure 1

cla-rified that primer 890 produced 15 total bands (three unique and three monomorphic), the remainder nine

poly-morphic bands reaching the polymorphism 80%, primer 1789 b generated 14 bands (one unique and four

mo-nomorphic) with 71.4% polymorphism ratio where the polymorphic bands were nine, 12 polymorphic bands

produced by primer 835 in addithion to one monomorphic and two unique bands the polymorphism reached

93.3%, no unique bands were generated by both primers 851 and 809 the former produced six monomorphic

bands and six polymorphic ones giving 60% polymorphism while the latter produced three monomorphic bands

and 14 polymorphic ones increasing the polymorphism to 82.3%. Coding of character states was produced in

Table 3

. The obtained cladogram (

Figure 2

) showed two series, the second one had two subseries, one of them

divided into two clusters, each one divided into two groups.

(a)

(b)

(c)

(d)

(e)

Figure 1.

ISSR profiles of the studied taxa of Acanthaceae senso lato generated by

Table 3.

Morphological & molecular characters (55), their states (172) and codes of taxa under investigation.

No. Character Character State and its (Code)

1 Duration Perennial (1), Annual (0)

2 Gross morphology Shrub: present (1) Absent (0), Subshrub: present (1) Absent (0), Herb: present (1) Absent (0)

3 habit Climbing (1), Erect (0)

4 Leaf composition Lobed (1), Simple (0)

5 Leaf arrangement Decussate (1), Superposed (0)

6 Leaf shape Lanceolate: present (1) Absent (0), Ovate: present (1) Absent (0), Obovate: present (1) Absent (0), Oblonovate: present (1) Absent (0), Cordate: present (1) Absent (0)

7 Leaf margin Entire: present (1) Absent (0), Spiny: present (1) Absent (0), Sinuate: present (1) Absent (0), Crenate: present (1) Absent (0)

8 Leaf apex Acute: present (1) Absent (0), Acuminate: present (1) Absent (0), Obtuse: present (1) Absent (0)

9 Leaf texture Glabrous: present (1) Absent (0), Hairy: present (1) Absent (0), Spiny: present (1) Absent (0)

10 Leaf base Cuneate: present (1) Absent (0), Rounded: present (1) Absent (0), Cordate: present (1) Absent (0), Truncate: present (1) Absent (0)

11 Bract Present (1) Absent (0)

12 Sepals number Five: present (1) Absent (0), Four: present (1) Absent (0), Numerous: present (1) Absent (0)

13 Sepals union United (1) Free (0)

14 Corolla shape 1-lipped: present (1) Absent (0), Bilabiate: present (1) Absent (0), Funnel: present (1) Absent (0)

15 Petals texture Hairy (1) Glabrous (0)

16 Corolla tube length Long (1) Short (0)

17 Stamens number two (1) Four (0)

18 Stamens fertility Sterile (1) Fertile (0)

19 Stamens length Exerted (1) Included (0)

20 Stamens symmetry Unequal (0) Equal (1)

21 Stamens texture Hairy (1) Glabrous (0)

22 Ovary texture Hairy (1) Glabrous (0)

23 Stigma shape Forked: present (1) Absent (0), Single: present (1) Absent (0), lobed: present (1) Absent (0)

24 Basal nectary disc present (1) Absent (0)

25 1ry _{vein category Basal actinodromus (1) Pinnate (0)}

26 2ry _{vein category}

Festooned semicraspedoromum: present (1) Absent (0), Semicraspidodromus: present (1) Absent (0), Festooned brochidodromus: present (1) Absent (0), Brochidodromus: present (1) Absent (0), Eucamptodromous: present (1) Absent (0), Weak brochidodromus: present (1) Absent (0)

27 2ry vein spacing Uniform: present (1) Absent (0), Irregular: present (1) Absent (0),

Decreasing toward base: present (1) Absent (0), Increase toward base: present (1) Absent (0)

28 2ry _{vein angle} Abruptdly increase to base: present (1) Absent (0), Uniform: present (1) Absent (0), Smoothly increase toward base: present (1) Absent (0), Smoothly decrease toward base: present (1) Absent (0)

29 Intersecondary veins Absent: present (1) Absent (0), Weak: present (1) Absent (0), strong: present (1) Absent (0)

30 3ry _{vein category}

Alternate percurrent: present (1) Absent (0), Random reticulate: present (1) Absent (0), Opposite percureent: present (1) Absent (0), Mixed opp/alte: present (1) Absent (0),

Regular polygonal reticulate: present (1) Absent (0)

Continued

32 3ry _{vein angle Obtuse: present (1) Absent (0), Acute: present (1) Absent (0), Perpendicular: present (1) Absent (0)}

33 4ry _{vein category} Alternate percurrent: present (1) Absent (0), Dichotomizing: present (1) Absent (0), Regular polygonal reticulate: present (1) Absent (0), Ill-developed: present (1) Absent (0)

34 5ry _{vein category} Dichotomizing: present (1) Absent (0), Regular polygonal reticulate: present (1) Absent (0), Ill-developed: present (1) Absent (0)

35 Aereolation Well developed: present (1) Absent (0), Moderately developed: present (1) Absent (0), lacking: present (1) Absent (0)

36 Free ending ultimate vein

Absent: present (1) Absent (0), Unbranched: present (1) Absent (0), 1-branched: present (1) Absent (0), 2-or more branched: present (1) Absent (0)

37 Marginal

ultimate venation Incomplete loop(1) Looped(0)

38 Abaxial Lamina

epidermal cell shape Irregular (1)Polygonal (0)

39 Abaxial Epidermal cell anticlinal wall

Straight: present (1) Absent (0), Undulate: present (1) Absent (0), Sinuous: present (1) Absent (0), Curved: present (1) Absent (0)

40 Abaxial Stomata type Diacytic: present (1) Absent (0), Amphidiacytic: present (1) Absent (0), Polocytic : present (1) Absent (0), Amphipericytic: present (1) Absent (0)

41 Abaxial Abnormal stomata Present (1) Absent (0)

42 Trichome type Absent: present (1) Absent (0), Non glandular uniseriate: present (1) Absent (0), Non glandular unicellular: present (1) Absent (0), Glandular: present (1) Absent (0)

43 Abaxial Trichome

foot cell Present (1) Absent (0)

44 Abaxial Crystals Present (1) Absent (0)

45 Leaf type Amphistomatic (1) Hypostomatic (0)

46 Lamina surface sculpture

Colliculate: present (1) Absent (0), Ruminate: present (1) Absent (0), Reticulate: present (1) Absent (0), Rugose: present (1) Absent (0).

47 Anticlinal wall width Broad (1) Narrow (0)

48 Anticlinal wall elevation Depressed (1) Raised (0)

49 Periclinal wall elevation Raised (1) Depressed (0)

50 Periclinal wall texture Striate (1) Smooth (0)

51 Primer 890 Bands 1 - 12: Present (1) Absent (0)

52 Primer 17898b Bands 1 - 10: Present (1) Absent (0)

53 Primer 835 Bands 1 - 14: Present (1) Absent (0)

54 Primer 851 Bands 1 - 6: Present (1) Absent (0)

55 Primer 809 Band 1 - 14: Present (1) Absent (0)

4. Discussion

The phenetic analysis of both morphological and molecular attributes generated a dendrogram that clarifies the

segregation of genus

Avicennia

as a distinct identity owing to three characters

viz

. the shrub habit, the presence

of moderately developed areolation and the ocellate adaxial surface sculpture. This is in accord with [6] who

used data from both the chloroplast and the nuclear genome, that implied the black mangrove genus

Avicennia

,

usually treated as a separate family in Lamiales or as a genus within Verbenaceae, but more recently has been

placed by some botanists in the monogeneric family Avicenniaceae and recent phylogenetic study [48] has

sug-gested that

Avicennia

is derived from Acanthaceae but is included in that family according to [49].

Figure 2.

UPGMA dendrogram based on 173 morphological and molecular attributes of the studied taxa of Acanthacea

sen-so lato.

Inside Series II,

Blepharis edulis

segregated as a distinct identity at a taxonomic distance less than 1.176

ow-ing to the incomplete looped marginal ultimate vein. The core of the present series distributed into two subseries

(A & B) at a taxonomic distance 1.176. In taxonomic context

Blepharis

and

Acanthus

are joined in the same

tribe Acantheae of most current taxonomic systems [2] [5] [10] [13] [14] [50] [51] the present study did not

support this combination, while the segregation of

Blepharis

from Series II at taxonomic distance near

Acanthus

group may maintain the combination of

Blepharis

with

Acanthus

in a single tribe as the previous taxonomic

systems.

Subseries A that marked with valuable taxonomic traits viz. the climbing habit and the numerous sepals,

in-cluded

Thunbergia alata

,

T. grandiflora

&

T. erecta

, this is in agreement with [10] [13] [14] who recorded

Thunbergia

in a tribe Thunbergiae while [2] [50] [51] recorded the same genus in a subfamily Thunbergioideae.

[52] reported that the Thunbergioideae comprised five genera,

Thunbergia

is the largest of them, and the

subfa-mily is characterized by a predominantly twining habit, enlarged bracteoles, and a reduced calyx. Furthermore

Thunbergioideae lack the retinaculate fruits found in core Acanthaceae.

Group II includes

Pseuderanthemum atropurpureum

,

P. bicolor

&

Sanchizia nobilis

,

Aphelandra squarossa

,

Odontonema cuspidatum

,

Anisacanthus virgularis

,

Justicia gendraussa

,

jacobinia carnea

,

J. specigera

,

Pa-chystachys lutea

,

J. adhatoda

,

J. betonica

&

J. brandegeeana

.

Pseuderanthemum atropurpureum

,

P. bicolor

&

Sanchizia nobilis

separated in a Subgroup A at a taxonomic

distance less than 1.008, owing to the presence of sterile stamens. This is in accord with [50], while in contrast

with [4] [5] that placed

Pseuderanthemum

and

Sanchizia

in two different tribes; Trichanthereae and Justicieae

respectively.

Genus

Odontonema

and

Aphelandra

separated away from the remaining members in a subgroup B in clan 1 at

a taxonomic distance less than 0.868 owing to the reticulate adaxial surface sculpture.

Justicia

species with

Ja-cobinia

and

Pachystachys

srparated in a Clan 2 at a taxonomic distance 1.036. The inclusion of

Pachystachys

with

Justicia

and

Jacobinia

have been recorded in tribe Gendarusseae according to [10], and in a tribe Justicieae

according to [4] [5], while [2] segregated

Pachystachys

in a single tribe named Graptophylleae. [50] combined

Eranthemum

,

Ruellia

&

Sanchizia

(subtribe Ruelliinae),

Anisacanthus

,

Fittonia

,

Hypoestes

,

Justicia

,

Odonto-nema

&

Pseuderanthemum

(Subtribe Justiciinae) and

Barleria

(Subtribe Barleriinae) all in a single tribe

Ruel-lieae.

5. Conclusion

Finally it is recommended that all of these hypotheses clearly merit additional study and need to be tested

against more data, including both more characters and more taxa. For precise delimitation and the membership

of them. In conclusion, the phenetic analysis of both morphological and molecular attributes clarified the

segre-gation of genus

Avicennia

as a distinct identity away from Acanthaceae.

Acanthus mollis

&

A. montanus

are

isolated in its own series that comparable to tribe Acantheae of the current taxonomic systems as the same as the

studied species of

Thunbergia

in its own subseries that comparable to tribe Thunbergiae and

Ruellia

in its own

group that comparable to tribe Ruellieae.

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[1]

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[2]

Lindau, G. (1895) Acanthacaeae. In: Engler, A. and Prantel, K.,

Die Naturalichen Pflanzenfamilien

, Vol. 4, 274-354.

[3]

Scotland, R.W., Sweere, J.A., Reeves, P.A. and Olmstead, R.G. (1995) Higher-Level Systematics of Acanthaceae

De-termined by Chloroplast DNA Sequences.

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Appendix

Table A1.

Macromorphological characters of the studied taxa.

C

T Duration

Gross

morphology Habit

Leaf composition

Leaf arrangement

Lamina shape

Lamina margin

Lamina apex

Lamina texture

Lamina base

1 Annual Herb Erect Lobed Decussate Lanceolate Sinuate Acute Glabrous Cuneate

2 Perennial ≈ ≈ ≈ ≈ ≈ Spiny Acuminate Spiny ≈

3 ≈ Sub-shrub ≈ Simple ≈ ≈ Entire ≈ Glabrous ≈

4 Annual Herb ≈ ≈ ≈ Obovate ≈ ≈ ≈ ≈

5 Perennial Sub-shrub ≈ ≈ ≈ Ovate ≈ ≈ Hairy ≈

6 ≈ ≈ ≈ ≈ ≈ ≈ ≈ acute ≈ ≈

7 ≈ Herb ≈ ≈ Superposed Lanceolate Spiny ≈ ≈ ≈

8 ≈ Sub-shrub ≈ ≈ Decussate Ovate Entire Acuminate Glabrous ≈

9 Annual Herb ≈ ≈ ≈ ≈ ≈ Acute Hairy Rounded

10 ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈

11 ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ Glabrous ≈

12 Perennial Sub-shrub ≈ ≈ ≈ Lanceolate ≈ ≈ ≈ Cuneate

13 ≈ ≈ ≈ ≈ ≈ Ovate ≈ Acuminate ≈ ≈

14 ≈ ≈ ≈ ≈ ≈ ≈ ≈ Acute ≈ ≈

15 ≈ Herb ≈ ≈ ≈ ≈ ≈ ≈ Hairy ≈

16 ≈ Sub-shrub ≈ ≈ ≈ Lanceolate ≈ ≈ Glabrous ≈

17 ≈ ≈ ≈ ≈ ≈ Oblongovate ≈ ≈ ≈ ≈

18 ≈ ≈ ≈ ≈ ≈ Ovate ≈ Acuminate ≈ ≈

19 ≈ ≈ ≈ ≈ ≈ Lanceolate ≈ ≈ ≈ ≈

20 ≈ ≈ ≈ ≈ ≈ Ovate ≈ Obtuse ≈ Rounded

21 ≈ ≈ ≈ ≈ ≈ Ovate ≈ Acute ≈ Cuneate

22 Annual Herb ≈ ≈ ≈ Ovate ≈ ≈ Hairy ≈

23 ≈ ≈ ≈ ≈ ≈ Lanceolate ≈ ≈ Glabrous ≈

24 ≈ ≈ ≈ ≈ ≈ Ovate ≈ ≈ Glabrous ≈

25 ≈ ≈ ≈ ≈ ≈ Ovate ≈ ≈ Hairy ≈

26 Perennial Sub-shrub ≈ ≈ ≈ Ovate Crenate Acuminate Glabrous ≈

27 ≈ ≈ Twining ≈ Superposed Cordate Sinuate Acute Hairy Cordate

28 ≈ ≈ ≈ ≈ ≈ Ovate Entire Acuminate Glabrous ≈

29 ≈ ≈ ≈ ≈ ≈ Ovate Sinuate ≈ Glabrous Truncate

C

T Bract

Sepal No.

Sepal union

Corolla shape

Corolla tex.

Corolla tube

No. of

stamens Staminodes L.R.C.

Stamens symmetry

Stamens tex.

Gynoecium tex.

Stigma

shape Disc

1 + 5 United 1-lipped G. Short 4 Absent Exerted Equal G. G. Forked -

2 ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈

3 ≈ ≈ ≈ Funnel ≈ Long 2 ≈ ≈ ≈ ≈ ≈ Single +

4 ≈ ≈ Free Bilabiate ≈ ≈ 4 ≈ Included ≈ ≈ ≈ Forked -

5 ≈ 4 United Funnel ≈ ≈ ≈ ≈ ≈ Unequal ≈ ≈ Single +

6 ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈

7 ≈ ≈ Free 1-lipped ≈ Short ≈ ≈ ≈ Equal ≈ ≈ ≈ ≈

8 ≈ 5 United Funnel ≈ Long ≈ ≈ Exerted ≈ ≈ ≈ Lobed -

9 ≈ ≈ ≈ ≈ ≈ Short 2 ≈ Included ≈ ≈ ≈ Single ≈

10 ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈

11 ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ +

12 ≈ ≈ ≈ Bilabiate ≈. Long ≈ ≈ Exerted ≈ ≈ ≈ ≈ ≈

13 ≈ ≈ ≈ ≈ H. Short ≈ ≈ ≈ ≈ ≈ H. Forked ≈

14 ≈ ≈ ≈ ≈ G. ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈

15 ≈ ≈ ≈ ≈ H. Long ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈

16 ≈ ≈ ≈ ≈ G. Short ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈

17 ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ Single ≈

18 ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ Forked ≈

19 ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈

20 ≈ ≈ ≈ Funnel ≈ Long ≈ 2 Included Unequal ≈ ≈ ≈ -

21 ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈

22 - ≈ ≈ ≈ ≈ ≈ 4 Absent ≈ Equal H. G. Single +

23 ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈

24 ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈

25 ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈

26 + ≈ ≈ Bilabiate ≈ Short 2 2 Exerted Unequal G. H. ≈ -

27 - ∞ Free Funnel ≈ Long 4 Absent ≈ ≈ H. G. ≈ +

28 ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈

29 ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈ ≈

Table A2.

Lamina epidermal characteristics of the studied taxa (LM).

Foot cell of Trichomes (Ab/Adaxial) Trichome type Abnormal stomata (Ab/Adaxial) Leaf type

Irregular/Same Sinuous/Curved Diacytic &

Amphidiacytic/- +/+ −/−

NG.

uniseriate +/− Hypostomatic ≈ Curved/Sinuous Diacytic & Polocytic/− −/− +/+ ≈ −/− ≈ ≈ Sinuous/Same Diacytic & polocytic/- +/+ −/− − ≈ ≈

Polygonal/Same Straight/Same Diacytic/- −/− ≈ NG.

uniseriate ≈ ≈

Irregular/Same Sinuous/Same Diacytic& polocytic/- ≈ ≈ ≈ ≈ ≈

≈ Undulate/Same Diacytic/- +/+ +/+ NG.

unicellular ≈ ≈

Polygonal/Same Straight/Sinuous Diacytic/Diacytic, _{Amphipericytic} +/− −/−

NG. unicellular,

glandular

≈ Amphistomatic

≈ Straight/Same Amphipericytic/- +/+ +/+ NG.

uniseriate ≈ Hypostomatic

≈ Straight/undulate Diacytic/- ≈ ≈ ≈ ≈ ≈

Irregular/Same Undulate/Sinuous ≈ −/+ ≈ ≈ ≈ ≈

≈ Undulate/Same ≈ +/+ −/−

NG. unicellular

glandular

+/− ≈

≈ Sinuous Same Diacytic, polocytic/- −/− ≈ NG.

uniseriate −/− ≈

≈ ≈ Diacytic/- ≈ +/− ≈ ≈ ≈

≈ Curved/Same Diacytic/Same ≈ −/− − ≈ Amphistomatic

≈ Undulate/Sinuous Diacytic/- +/− ≈ NG.

uniseriate ≈ Hypostomatic

≈ Undulate/Curved ≈ −/+ +/+ − ≈ ≈

≈ Sinuous/Same Diacytic& polocytic/- −/− −/+ NG.

uniseriate ≈ ≈

Polygonal/Same Straight/Same Diacytic &

amphidiacytic/- ≈ −/− ≈ ≈ ≈

Irregular/Same Undulate/Curved Diacytic/Same +/+ +/+

NG. uniseriate,

glandular

≈ Amphistomatic

Polygonal/Same Straight/Same Diacytic/- −/+ −/+ − ≈ Hypostomatic

≈ ≈ ≈ −/− +/− ≈ ≈ ≈

Irregular/Same Curved/Undulate ≈ ≈ −/−

NG. uniseriate,

glandular

≈ ≈

Polygonal/Same Straight/Same Diacytic,

Amphipericytic/Diacytic ≈ ≈ − ≈ Amphistomatic

≈ Curved/Same Diacytic/Same ≈ ≈ ≈ ≈ Amphistomatic

Irregular/Same ≈ ≈ +/+ ≈ ≈ +/− Amphistomatic

Polygonal/Same Straight/Same Amphipericytic/same +/− +/+ ≈ −/− ≈

Irregular/Same Curved/Sinuous Diacytic/Same −/− −/−

NG. uniseriate,

NG. unicellular

≈ ≈

≈ Sinuous/Same Diacytic/- ≈ ≈ − ≈ Hypostomatic

≈ Curved/Same ≈ +/− ≈ NG.

unicellular ≈ ≈

Polygonal/Same Curved/Straight ≈ −/− ≈ − ≈ ≈

Table A3.

Lamina epidermal characteristics of the studied taxa (abaxial surface, SEM).

C

T Sculpture

Anticlinal wall width

Anticlinal wall elevation

Anticlinal wall surface

Periclinal wall elevation

Periclinal wall surface

Ruminate Broad Depressed Smooth Raised Smooth

Colliculate Narrow ≈ ≈ ≈ ≈

Ruminate ≈ ≈ ≈ ≈ ≈

Reticulate ≈ Raised ≈ Depressed ≈

≈ ≈ ≈ ≈ ≈ Striate

Colliculate ≈ Depressed ≈ Raised Smooth

Ruminate ≈ ≈ ≈ ≈ Striate

Reticulate ≈ Raised ≈ Depressed ≈

Rugose ≈ ≈ ≈ ≈ ≈

≈ ≈ ≈ ≈ ≈ ≈

Reticulate ≈ ≈ ≈ ≈ ≈

Rugose ≈ ≈ ≈ ≈ Smooth

Ruminate Broad Depressed ≈ Raised ≈

≈ ≈ ≈ ≈ ≈ ≈

≈ ≈ ≈ ≈ ≈ Striate

≈ ≈ ≈ ≈ ≈ Smooth

Rugose Narrow Raised ≈ Depressed ≈

Reticulate ≈ ≈ ≈ ≈ ≈

Rugose ≈ ≈ ≈ ≈ ≈

Ruminate ≈ Depressed ≈ Raised ≈

≈ ≈ ≈ ≈ ≈ ≈

Rugose ≈ Raised ≈ Depressed ≈

Reticulate ≈ ≈ ≈ ≈ ≈

Rugose ≈ ≈ ≈ ≈ ≈

Ruminate ≈ Depressed ≈ Raised ≈

≈ ≈ ≈ ≈ ≈ ≈

≈ ≈ ≈ ≈ ≈ ≈

≈ ≈ ≈ ≈ ≈ ≈

≈ ≈ ≈ ≈ ≈ ≈

Table A4.

Lamina vein architecture of the studied taxa.

spacing 2 vein angle

Inter 2 veins 3 vein category 3 vein course 3 vein angle to 1

Pinnate Festooned

semicraspedoromus Irregular

Abruptdly

increase to base Weak

Alternate percurrent

Exmedially

ramified Acute

≈ Semicraspidodromus Uniform ≈ ≈

Regular polygonal

reticulate

≈ Obtuse

≈ _{brochidodromus} Festooned _{toward base} Decrease Uniform ≈ Random reticulate

Admedially

ramified ≈

≈ ≈ Uniform Smoothly

increase to base Absent Mixed ≈ ≈

≈ ≈ _{toward base} Decrease Uniform Strong ≈ ≈ ≈

≈ ≈ ≈ ≈ Absent ≈ ≈ ≈

≈ Semicraspidodroms Irregular ≈ ≈ Random

reticulate ≈ Acute

≈ Brochidodromus Decrease

toward base Uniform ≈ Mixed Straight Obtuse

≈ ≈ ≈ Smoothly increase toward base

≈ _percurent Opposite Sinuous ≈

≈ _{brochidodromus} Festooned ≈ ≈ ≈ Mixed Admedially

ramified ≈

≈ ≈ Irregular ≈ ≈ Alternate

percurrent ≈ ≈

≈ ≈ _{toward base} Decrease Uniform ≈ opposite

percurrent Straight Perpendicular

≈ ≈ ≈ ≈ ≈ Mixed Sinuous Obtuse

≈ ≈ ≈ Smoothly decrease

toward base

Weak Alternate percurrent

Admedially

ramified ≈

≈ ≈ ≈ Uniform Absent Random

reticulate ≈ Acute

≈ Eucamptodromous ≈ ≈ Weak ≈ ≈ ≈

≈ _{brochidodromus} Festooned Uniform ≈ Absent Mixed Straight Obtuse

≈ ≈ _{toward base} Decrease Smoothly increase toward base

Strong _reticulate Random Admedially _ramified ≈

≈ _{brochidodromus} Weak ≈ Uniform Weak opposite

percurrent Straight ≈

≈ _{brochidodromus} Festooned Uniform

Smoothly increase toward base

Absent Mixed Sinuous ≈

≈ ≈ ≈ Uniform Weak Random

reticulate

Admedially

ramified ≈

≈ Weak brochidodromus Decrease

toward base ≈ Absent ≈ ≈ ≈

≈ ≈ ≈ ≈ ≈ polygonal Regular

reticulate

≈ Perpendicular

Continued

≈ _{brochidodromus} Weak _{toward base} Increase ≈ ≈ ≈ ≈ ≈

≈ _{brochidodromus} Festooned ≈ ≈ ≈ ≈ ≈ Acute

Basal

actiondromus ≈ ≈

Abruptdly

increase to base Weak ≈ ≈ Obtuse

Pinnate ≈ Uniform Uniform Absent ≈ ≈ ≈

Basal

actinodromus Brochidodromus

increase toward base

Abruptdly

increase to base Weak Mixed ≈ Perpendicular

Pinnate Brochidodromus ≈ Uniform ≈ ≈ ≈ Obtuse

C

T 4 vein category 5 vein category Areolation F. E. V. S Mar. veins

Regular polygonal reticulate Dichotomizing Well developed 2 or more branched Looped

Dichotomizing Ill developed ≈ Absent ≈

Alternate percurrent Dichotomizing ≈ 2 or more branched ≈

Regular polygonal reticulate Ill developed Lacking Absent ≈

Alternate percurrent ≈ Well developed ≈ ≈

≈ Dichotomizing ≈ 2 or more branched ≈

Regular polygonal reticulate Ill developed ≈ ≈ Incomplete loop

≈ ≈ ≈ Unbranched Looped

Ill developed ≈ ≈ Absent ≈

Alternate percurrent ≈ ≈ 2 or more branched ≈

≈ ≈ ≈ ≈ ≈

≈ Regular polygonal reticulate ≈ ≈ ≈

≈ ≈ ≈ ≈ ≈

Regular polygonal reticulate Ill developed ≈ ≈ ≈

≈ ≈ ≈ ≈ ≈

≈ Ill developed ≈ 1-branched ≈

Alternate percurrent Dichotomizing ≈ 2 or more branched ≈

Regular polygonal reticulate Ill developed ≈ Absent ≈

≈ Dichotomizing ≈ 2 or more branched ≈

≈ Ill developed ≈ Unbranched ≈

≈ Dichotomizing ≈ 2 or more branched ≈

≈ Regular polygonal reticulate ≈ Absent ≈

≈ Ill developed ≈ 2 or more branched ≈

≈ Dichotomizing ≈ Unbranched ≈

≈ Ill developed ≈ Absent ≈

≈ Dichotomizing ≈ 2 or more branched ≈

≈ Ill developed ≈ Absent ≈

≈ Dichotomizing ≈ 2 or more branched ≈

≈ ≈ ≈ ≈ ≈